DNA ligation

Added By

Ligation Mix

• Example - 10ul mix

• 1.0 [math]\displaystyle{ \mu }[/math]L 10X T4 ligase buffer

• 0.5 [math]\displaystyle{ \mu }[/math]L T4 Ligase

• 6:1 Molar ratio of insert to vector ([math]\displaystyle{ \sim }[/math]10ng vector)
• X[math]\displaystyle{ \mu }[/math]L dd[math]\displaystyle{ \rm{H_2O} }[/math] to bring the total to 10[math]\displaystyle{ \mu }[/math]L

Calculating Insert and Vector Amounts

Insert Mass =[math]\displaystyle{ 6\times\left[\frac{\rm{Insert\ Length}}{\rm{Vector\ Length}}\right]\times \rm{Vector\ Mass} }[/math]

Reaction Conditions

• Let the 10 [math]\displaystyle{ \mu }[/math]L solution sit at [math]\displaystyle{ 22.5^o }[/math]C for 30min
• Denature the ligase at [math]\displaystyle{ 65^o }[/math]C for 10min
• Dialyze for 20 minutes if electroporating
• Use disks shiny side up
• Store at -20C